Techniques for accelerating thermal equilibrium in a chromatographic column

ABSTRACT

Techniques are described for accelerating thermal equilibrium in a chromatographic column. An apparatus comprises a chromatography column, and a plurality of temperature control units in thermal contact with the chromatography column. A method of performing liquid chromatography comprises setting an inlet of a chromatography column to a first temperature using a first temperature control unit in thermal contact with said inlet, setting an outlet of the chromatography column to a second temperature using a second temperature control unit in thermal contact with the outlet, wherein the first temperature is less than the second temperature; and injecting a sample into a liquid stream that flows through the chromatography column after the inlet is set at the first temperature and the outlet is at the second temperature.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/406,285, filed Dec. 8, 2014, now U.S. Pat. No. 10,478,750, which is anational phase filing of International Patent Application No.PCT/US2013/047804, filed on Jun. 26, 2013, which claims priority to andthe benefit of U.S. provisional application No. 61/668,565, filed Jul.6, 2012, entitled TECHNOQUES FOR ACCELERATING THERMAL EQUILIBRIUM IN ACHROMATOGRAPHIC COLUMN. The entire contents of these applications areincorporated herein by reference.

TECHNICAL FIELD

This application generally relates to techniques for use with liquidchromatography, and more particularly to thermally insulating a liquidchromatography column.

BACKGROUND INFORMATION

Chromatography is a technique for separating compounds, such as thoseheld in solution, where the compounds will exhibit different affinityfor a separation medium in contact with the solution. As the solutionflows through such an immobile separation medium, the compounds separatefrom one another. Common chromatographic separation instruments includeliquid chromatography (LC) systems. Such LC systems are known and usedfor analyzing sample solutions that may include different chemicalcompounds. LC systems may operate at high pressures such as at 5,000 PSIand greater. A typical LC system includes a pump for pumping a liquidsolution, an injector for injecting the sample into the liquid fluidstream, a chromatography column packed with packing material used as theseparation medium and tubing for carrying the sample solution and liquidfluid from the injector to the chromatography column. The tubing maythen be used to further transport the sample solution output from the LCcolumn to a detector for analyzing the sample solution. The detector maybe any suitable detector such as a mass spectrometer, a UV absorbancedetector, an evaporative light scattering detector, and the like.

During operation of the LC system, a liquid solvent is pumped into theLC system at high pressure. The injector may be used to injectcontrolled volumes of a sample, either manually or automatically, intothe system where the sample is carried with the liquid solvent in afluid stream to the packed LC column where the sample may then beseparated. Because each of the chemical compounds in the sample solutionreact in a different manner with the LC column packing material, thevarious chemical compounds flow through the packed LC column atdifferent rates. The different chemical compounds in the sample solutionseparate from each other as the sample solution flows through thecolumn. The separated chemical compounds as output from the LC columnproceed to the detector where they may be further analyzed, for example,to determine physical characteristics of the compounds for purposes ofidentification and/or quantification.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention is an apparatuscomprising: a chromatography column; and a plurality of temperaturecontrol units in thermal contact with the chromatography column. Each ofthe plurality of temperature control units may perform any of heatingand cooling. A first of the plurality of temperature control units maybe in thermal contact with an outlet of the chromatography column. Theoutlet may be at an end of the chromatography column out of which amobile phase exits the chromatography column. A first of the pluralityof temperature control units may be in thermal contact with an inlet ofthe chromatography column. The inlet may be at an end of thechromatography column into which a mobile phase flows. Each of theplurality of temperature control units may be in thermal contact withthe chromatography column at a different axial position of thechromatography column. The axial position may be a location with respectto an axis of flow for mobile phase through the chromatography column.The first of the plurality of temperature control units may be used toheat the inlet of the chromatography column to a first temperatureT_(in) and a second of the plurality of temperature control units may beused to heat an outlet of the chromatography column to a secondtemperature T_(out). The outlet may be located at an end of thechromatography column opposite the inlet and may be an end out of whichthe mobile phase exits the chromatography column. T_(in) may be lessthan T_(out), and T_(in) and T_(out) may both be greater than ambienttemperature. The first of the plurality of temperature control units maybe used to cool the inlet of the chromatography column to a firsttemperature T_(in) and a second of the plurality of temperature controlunits may be used to heat an outlet of the chromatography column to asecond temperature T_(out). The outlet may be located at an end of thechromatography column opposite the inlet and may be an end out of whichthe mobile phase exits the chromatography column. T_(in) may be lessthan T_(out) and T_(in) may be less than ambient temperature and T_(out)may be greater than ambient temperature. An insulating layer maysurround the chromatographic column. The insulating layer may be madefrom any of polystyrene foam, a polymer, polymethacrylate, silicone,urethane, polyolefins, polyamide, polysulfone, polyethyramide,polycarbonate, rubber, polyester, polyfluoroelastomers, polyethyleneterephthalate, a ceramic, an aerogel, a fibrous material,methylcellulose and fiberglass. The insulating layer may be formed in anarea between a jacket surrounding the chromatographic column and thechromatographic column. The insulating layer may comprise a vacuum. Thevacuum may comprise an inert gas. The inert gas may be any of argon,xenon, krypton, carbon dioxide and sulfur hexafluoride. The vacuum maycomprise atmospheric gas. The insulating layer and the jacket may be anintegral component of the chromatographic column. The insulating layermay comprise a chamber including aerogel particles. A first of theplurality of control units may be located at an inlet of a fluid paththrough the chromatographic column. A second of the plurality of controlunits may be located at an outlet of the fluid path through thechromatographic column. A first temperature sensor may sense a firsttemperature at the inlet. A second temperature sensor may sense a secondtemperature at the outlet. A control means may control adjustment of thefirst control unit to set the first temperature at the inlet to a firstdesired value and may control adjustment of the second control unit toset the second temperature to a second desired value. The differencebetween the first desired value and the second desired value mayrepresent a temperature difference, ΔT_(L), determined as

${\Delta\; T_{L}} = {\left( {1 - \overset{\_}{\alpha\; T}} \right)\frac{\Delta\; P}{\rho\; C_{p}}}$where α is a thermal expansion coefficient of the mobile phase, T is themean temperature of the mobile phase, ΔP is a pressure drop across thechromatographic column, C_(p) is a heat capacity of the mobile phase atconstant pressure, and αT represents the average of a quantity αT, and ρis a density of the mobile phase.

In accordance with another aspect of the invention is a method ofperforming liquid chromatography comprising: setting an inlet of achromatography column to a first temperature using a first temperaturecontrol unit in thermal contact with said inlet; setting an outlet ofthe chromatography column to a second temperature using a secondtemperature control unit in thermal contact with said outlet, whereinthe first temperature is less than the second temperature; and injectinga sample into a liquid stream that flows through the chromatographycolumn after the inlet is set at the first temperature and the outlet isat the second temperature. The chromatography column may be thermallyinsulated. The method may include monitoring temperatures at the inletand the outlet to determine when the inlet is at the first temperatureand when the outlet is at the second temperature to thereby determinethat the chromatography column is at steady state with respect to columntemperature. Each of the first temperature control unit and the secondtemperature control unit may perform any of heating and cooling. Thefirst temperature may be less than ambient temperature and the secondtemperature may be greater than ambient temperature. The firsttemperature and the second temperature may be greater than ambienttemperature. The first temperature and the second temperature may beless than ambient temperature. An intermediate point may be located onthe chromatography column between the inlet and the outlet and themethod may include setting the intermediate point of the chromatographycolumn to a third temperature using a third temperature control unitprior to injecting said sample, wherein the third temperature may bebetween the first temperature and the second temperature. The thirdtemperature may be determined as a sum of the first temperature and anapproximated value. The approximated value may be determined as atemperature offset proportional to a distance of the intermediate pointfrom the inlet. The third temperature may be determined as an offsetfrom the second temperature. The offset may be an approximated valuedetermined as a temperature offset proportional to a distance of theintermediate point from the outlet. A difference between the firsttemperature and the second temperature ΔT_(L) may be determined based on

${\Delta\; T_{L}} = {\left( {1 - \overset{\_}{\alpha\; T}} \right)\frac{\Delta\; P}{\rho\; C_{p}}}$where α is a thermal expansion coefficient of the mobile phase, T is themean temperature of the mobile phase, ΔP is a pressure drop across thechromatographic column, C_(p) is a heat capacity of the mobile phase atconstant pressure, and αT represents mean value of quantity αT, and ρ isa density of the mobile phase.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention.

FIG. 1 is an example of a system that may utilize the chromatographiccolumn embodiments described in accordance with techniques herein;

FIGS. 2-4 and 8-10 are examples of various chromatographic columnembodiments in accordance with techniques herein;

FIGS. 5A, 5B, 6 and 7 are examples of various chromatographic columnembodiments and other components in accordance with techniques herein;and

FIG. 11 is an example illustrating chromatograms from test resultsperformed by the inventors in accordance with testing differentenvironments.

DESCRIPTION

Referring to FIG. 1, shown is an embodiment of a system in accordancewith techniques herein. The system 100 may include components such asanalytical instruments for performing sample analysis. In oneembodiment, the system 100 may be an LC instrument system including aliquid chromatograph (LC) 104, detector 112, storage 114, and computer116. As will be described in following paragraphs, the system 100 may beused to perform analysis of sample 102 for detecting one or morecompounds of interest. The LC 104 may include an injector 106 thatreceives sample 102, a pump 108, and a column 110. The liquid sample 102may be introduced as an input to the LC 104. Although not illustrated inFIG. 1, the LC 104 may also include an optional column heater. Asdescribed in more detail below, the computer 116 may be used to controloperation of the components and used in connection with data acquisitionto store analysis data to storage 114. As also described in more detailbelow, the sample and mobile phase traverse through the fluidic path ofthe system.

In operation, the sample 102 is injected into the LC 104 via theinjector 106. The pump 108 pumps the sample through the column 110 toseparate the sample into component parts according to retention timethrough the column 110. A high pressure stream of chromatographicsolvent provided by pump 108 and injector 106 forces sample 102 tomigrate through a chromatographic column 110 in the LC 104. Column 110typically comprises a packed column of porous, non-porous orsuperficially-porous particles, made of silica, polymer, or anorganohybrid silica whose surface may be chemically modified. The outputfrom the column 110 is input to the detector for analysis. The detector112 may be any suitable detector such as a UV absorbance detector, anevaporative light scattering detector, a mass spectrometer, and thelike.

In one embodiment, the LC system may be, for example, a High PerformanceLiquid Chromatography (HPLC) or an Ultra Performance LiquidChromatography (UPLC) system such as the ACQUITY UPLC and nanoACQUITYUPLC® systems from Waters Corporation of Milford Mass. An LC system suchas the foregoing from Waters Corporation may operate under high pressuresuch as in the range of 5000 PSI (e.g, exemplary for some HPLC systems)to 15000 PSI (exemplary for some UPLC systems).

A control means (not shown) provides control signals for the variouspower supplies (not shown) which respectively provide the necessaryoperating potentials for the components of the system 100 such as the104 and 112. These control signals determine the operating parameters ofthe instrument. The control means is typically controlled by signalsfrom a computer or processor, such as the computer 116.

The storage 114 may be any one or more different types of computerstorage media and/or devices. As will be appreciated by those skilled inthe art, the storage 114 may be any type of computer-readable mediumhaving any one of a variety of different forms including volatile andnonvolatile, removable and non-removable media implemented in any methodor technology for storage of information such as computer readableinstructions, data structures, program modules or other data. Computerstorage media includes, but is not limited to, RAM, ROM, EEPROM, flashmemory or other memory technology, CD-ROM, (DVD) or other opticalstorage, magnetic cassettes, magnetic tape, magnetic disk storage orother magnetic storage devices, or any other medium which can be used tostore the desired code, data, and the like, which can accessed by acomputer processor.

The computer 116 may be any commercially available or proprietarycomputer system, processor board, ASIC (application specific integratedcircuit), or other component which includes a processor configured toexecute code stored on a computer readable medium. The processor, whenexecuting the code, may cause the computer system 116 to performprocessing steps such as to access and analyze the data stored onstorage 114. The computer system, processor board, and the like, may bemore generally referred to as a computing device. The computing devicemay also include, or otherwise be configured to access, a computerreadable medium, such as represented by 114, comprising executable codestored thereon which cause a computer processor to perform processingsteps.

One or more molecular species migrates through column 110 and eachemerges, or elutes, from column 110 and is detected by detector 112. Thetime that it takes for a molecule to traverse through the column iscommonly is referred to as the molecule's retention time. That is, amolecule that elutes from a column at retention time t in reality elutesover a period of time that is essentially centered at time t. Theelution profile over the time period is referred to as a chromatographicpeak whereby the retention time for the molecule corresponds to the apexof the profile. The elution profile of a typically well-behavedchromatographic peak can be described by a Normal (Gaussian)distribution. The peak has a width that typically is described by itsfull width at half height, or half-maximum (FWHM).

The retention time and chromatographic peak profile of a moleculeeluting from a chromatographic support matrix (e.g., such packingmaterial of column 110 or other separation medium for separatingchemical compounds of a sample solution) is a function of the physicaland chemical interaction of that molecule between the support matrix andmobile phase. The degree of interaction that a molecule has between thesupport matrix and the mobile phase dictates the chromatographic profileand retention time for that molecule. In a complex mixture, eachmolecule is chemically different. As a result, each molecule can have adifferent affinity for the chromatographic matrix and the mobile phase.Consequently, each can exhibit a unique chromatographic profile.

When the sample solution flows through the packed LC column at highpressures, frictional heat is generated within the column. The amount offrictional heat generated is a function of several factors such as, forexample, the flow rate of the mobile phase, the particle size of thecolumn packing material, and the dimensions (length and inner diameter)of the column. Such frictional heat may result in an increase ordifference in the temperature at the center of the column relative tothe outer edges or walls of the column thereby causing a radial thermalgradient which adversely affects the performance of the LC system. Asknown in the art, LC performance may be measured, for example, in termsof efficiency by plate count, reduced plate height, and/or tailingfactors. Adversely affecting LC performance may be observed, forexample, by having a wider than normal/expected peak width, asymmetricalpeak shape, reduced plate count, and the like. For example, the radialthermal gradient, where the temperature at the column center is greaterthan at the column outer edges, causes the liquid mobile phase passingthrough the center to have a lower viscosity than at the outer edges. Asa result, the liquid mobile phase flows faster through the column centerthan at the outer edges. Also, since chromatographic retention typicallydecreases as temperature increases, an analyte migrates faster at thecenter of the column. To further illustrate, for example,chromatographic peaks may be broader or wider due to such changes inviscosity (and thus flow rate) due to radial thermal gradients in thecolumn as just described. Such increased peak widths may result inoverlapping peaks thereby adversely affecting the quality of theinformation obtained from LC data. Therefore, due to such adverseeffects (the foregoing of which is one example), it is desirable tominimize or reduce radial thermal gradients to improve LC performance.

Additionally, such frictional heat may cause a temperature differencewith respect to the direction of flow (also referred to as the axialdirection with respect to the axis of directional flow) through the LCcolumn. Such a temperature difference in the axial direction may bereferred to as an axial thermal gradient and may be measured bydetermining a temperature T_(in) of the liquid mobile phase enteringinto the LC column and a temperature T_(out) when exiting the LC column.When the particle size of packing in the LC column is, for example, a 5micron particle, there may be little difference between T_(in) andT_(out) in the axial direction (e.g., may be 1 or 2 degrees C.).However, with smaller size particles comprising the LC column packingsuch as particles having a size of 1.7 microns, the difference betweenT_(in) and T_(out) in the axial direction is much larger in comparisonto the axial thermal gradient for the 5 micron size particle case. Theaxial thermal gradient may affect retention but may have a minimal orinsignificant adverse impact on LC performance.

Generally, any type of temperature gradients with respect to the LCcolumn, (e.g., including any of radial and axial thermal gradients), mayhave an effect upon mobile phase viscosity, the speed or rate at whichan analyte in mobile phase diffuses, and may also affect retentivity(e.g., how an analyte interacts with the surfaces of the particles inthe column packing) thereby affecting chromatographic retention time. Asnoted above, an axial thermal gradient generally does not have asignificant negative or adverse affect on chromatographic performance.However, existence of a radial thermal gradient typically does have asignificant adverse impact on LC performance such as may be measured interms of column efficiency.

Through modeling and general testing of column environments includingadiabatic, isothermal and ambient, it has been determined that having acolumn environment of adiabatic (or that which approaches adiabaticconditions as close as possible) is best in efforts to have the smallestor minimal radial thermal gradient and the highest column efficiency(e.g., such as may be measured in terms of USP Plate Counts and/or HETP(Height Equivalent to the Theoretical Plate). Isothermal may be definedas having a constant column temperature at the column outer wall suchas, for example, by placing the column in a water bath. Adiabatic may bedefined as providing an outer insulation to the column to thereby reduceor eliminate any addition or removal or heat from the column. Ambientmay be defined as having the column in still air.

To this end, described herein are embodiments on the LC column whichprovide for such adiabatic conditions and minimize radial thermalgradients to maximize column efficiency and performance. Duringoperation of the LC system with such a column as described herein withthe goal of providing adiabatic conditions, the column heats upnaturally via friction as described elsewhere herein. At some point, thecolumn and LC system will reach a steady state temperature. Such asteady state with respect to column temperature may be determined byhaving T_(in), T_(out), and the difference therebetween (e.g., axialtemperature gradient) be relatively or substantially constant. In thissteady state with respect to column temperature (as may be determined byobtaining substantially constant values for T_(in), T_(out) and theaxial thermal gradient and with adiabatic conditions using the insulatedcolumn described herein), the temperature at the column outer wall andat the column center will be substantially the same thereby minimizingor eliminating the radial thermal gradient. Therefore, in such a steadystate temperature using the column embodiments described in more detailherein providing adiabatic conditions via column insulation, there willbe an axial thermal gradient and minimal radial thermal gradient.

It will be appreciated by those of ordinary skill in the art that the LCsystem may reach steady state prior to injecting a sample in connectionwith performing an LC experiment.

Referring to FIG. 2, shown is an example of an embodiment of achromatography column in accordance with techniques herein. Theembodiment of FIG. 2 may be used as the column 110 in the system ofFIG. 1. FIG. 2 illustrates a side cutaway view of a high performanceliquid chromatography (HPLC) column 128 within an insulating layer ormember 130 formed between the column 128 and an outer jacket 120. Inlettube 10 carries sample solution into the HPLC column 128 and outlet tube20 carries sample solution out of the HPLC column 128. Element 30represents the chromatographic separation medium, such as beads or othercolumn packing material. The insulating layer or member 130 may providethermal insulation for the HPLC column capable of operating at pressuresin excess of 5,000 PSI. In the embodiment of 125, the insulation layeror member 130 may be a vacuum chamber acting as a thermal insulator andhaving a pressure that is below local atmospheric pressure (e.g., belowthe atmospheric pressure external to the area 120 or surrounding thejacket 120). Examples of more specific pressure(s) that may be used inan embodiment in connection with the vacuum within the area 130surrounding the column are provided elsewhere herein. As illustrated,the column 128 may be placed within jacket 120 having suitable airtightscaling to isolate the column 128 (along with other illustratedcomponents attached thereto) located within the jacket 120 from theenvironment outside the jacket 120. Once the column 128 is placed withinthe jacket 120, a vacuum may be created in space or chamber 130 bypumping out the air therein surrounding the column 128 within thejacket. The air may be pumped out by connecting a vacuum pump (notillustrated) to through hole 50 of the jacket 120. Once the air has beenevacuated from the space 130, the through hole 50 may be sealed offthereby creating a vacuum chamber or space represented by 130 in thearea surrounding the column 128 within the jacket 120.

As a first alternative to the foregoing in which a vacuum chamber iscreated in space 130, the air in space 130 may be displaced with a heavyinert gas such as argon, krypton or xenon. In such an embodiment, asource of the inert gas (not illustrated) may be connected to throughhole 50 to displace the atmospheric gas of space 130 with the inert gas.The through hole 50 may then be closed off using any suitable means asdescribed above to form an air tight seal. In this first alternative,the insulating layer 130 may be formed by the inert gas located therein.As yet a further variation to the above prior to closing off throughhole 50, once the inert gas is provided in space 130, a vacuum pump maythen be attached to through hole 50 to create a vacuum by pumping outthe inert gas. Once a sufficient vacuum has been created, the throughhole 50 may then be closed off as described above to form a suitable airtight seal.

Thus, based on the foregoing exemplary alternatives, an embodiment mayform the insulating layer surrounding the column by pumping out the airor atmospheric gas in the space surrounding the column thereby creatinga vacuum chamber (with minimal atmospheric gas) as the insulating layer130. An embodiment may also form the insulating layer surrounding thecolumn by replacing the air or atmospheric gas in the space surroundingthe column with an inert gas thereby creating an inert gas chamber orlayer as the insulating layer 130 which is at atmospheric pressure. Anembodiment may also form the insulating layer surrounding the column bydisplacing the air or atmospheric gas in the space surrounding thecolumn with an inert gas and then pumping out the inert gas in the spacesurrounding the column thereby creating a vacuum chamber (with minimalinert gas) as the insulating layer 130.

In connection with the vacuum that may be formed in the chamber of theinsulating layer 130, it should be noted that a true vacuum has thelowest thermal conductivity whereby heat can only be transported byradiative heating. At very low pressure (e.g., approximately less than10⁻³ atm), thermal conductivity is directly proportional to pressure.This region is also known in the art as the Knudsen domain, where themean free path of the molecules is large compared to the dimensions ofthe chamber. At low pressures such as, for example, in the approximaterange of 10⁻³ atm to 10 atm, thermal conductivity is a very weakfunction of pressure, increasing less than ˜1% per bar. Thus, someembodiments may utilize a vacuum having a preferred pressure ofapproximately equal to, or less than, 10⁻³ atm. Other embodiments usingtechniques herein may utilize other pressures such as in the approximaterange of 10³ atm to 10 atm although pressures of approximately 10⁻³ atmor less may be preferred.

With respect to the gas that may be used in connection with 130 inembodiments described herein such as in the vacuum chamber at one of thepressures described herein, it should be noted that heavy gases providebetter thermal insulation than lighter gases, as they have lower thermalconductivity. Thermal conductivity in general decreases as molecularweight increases. An embodiment may use, for example, argon, xenon,and/or krypton which are much heavier than air, and thus have lowerthermal conductivity. As another example, an embodiment may forminsulating layer 130 using a gas including sulfur hexafluoride.

It should be noted that the column inner diameter may be any suitabledimension, such as 1 mm (millimeter) or greater although the increasedbenefits from using such a column may be more apparent as the columninner diameter increases (e.g., for example at inner column diameters of2 mm and greater). An embodiment may also use columns having anyparticle size for the packing material or, more generally, separationmedium. However, columns using smaller particle sizes, (e.g., such asparticles having a size of 2.5 microns or smaller) may typically obtaingreater benefits because more frictional heat is generated therebyresulting in larger thermal gradients. The outer column wall of column128 may be made from steel, titanium, or other suitable material able towithstand the HPLC operating pressures such as generally in excess of5,000 PSI. The surrounding jacket 120 may be made from steel or othersuitable material into which a through hole 50 may be machined orotherwise formed. The through hole 50 may be scaled in any suitablemanner, such as by crimping, capping (e.g., using a removable orpermanent cap), and the like, to thereby providing an airtight seal foruse in creating the vacuum in space represented by 130. For example, thethrough hole 50 may be sealed by having a removable cap (e.g. viathreading) applied thereto.

In one embodiment, the area 130 may form an insulating layer or memberand provide sufficient insulation preventing thermal conductivitybetween the column 128 and ambient temperature such as of theenvironment outside of or surrounding the jacket 120. The area denotedas 130 forming the insulating layer or member may provide thermalconductivity, for example, that is approximately at or below 0.02 W/mK.It should be noted that ideally it is desirable for the thermalconductivity provided by 130 to be less than that of air so that, forexample, an embodiment may use such gases denoted below having thermalconductivity less than that of air. (e.g., carbon dioxide, argon,krypton, xenon, sulfur hexafluoride). Below is a table of thermalconductivities of some gases at 1 atmosphere, 298 degrees K:

Compound Thermal Conductivity (W/mK) Helium 0.147 Hydrogen 0.175 Neon0.0483 Nitrogen 0.0255 Oxygen 0.0263 air 0.0259 Carbon dioxide 0.0169Argon 0.0174 Sulfur hexafluoride 0.0121 Krypton 0.00943 Xenon 0.00565

In connection with the above-reference information for the gases, suchinformation is generally available and known in the art. For example,data for all but Krypton, Xenon, and Sulfur hexafluoride (SF6), and airmay be obtained from RC Reid, J M Prausnitz, B E Poling, The Propertiesof Gases & Liquids, 4th Edition, McGraw Hill, 1987. Data for Krypton,Xenon, and Sulfur hexafluoride (SF6), and air may be found, throughpublically available information in the internet, for example, usingWolfram Alpha™ computation knowledge engine Wolfram Alpha LLC availableonline at www.wolframalpha.com.

The jacket 120 used in an embodiment may generally be any suitablematerial that can withstand a vacuum and does not outgas. For example,the jacket 120 may be made from one or more of steel, copper, brass,aluminum or other metals. The column may have walls constructed of, forexample, steel or titanium, but, more generally, may be made from anymaterial that can withstand the high pressures and also be chemicallyinert to a mobile phase and sample utilized in an embodiment. As analternative, an embodiment may select the column to have wallsconstructed from a material that does chemically interact with sample(e.g., ceramics will often interact with certain analytes), andcoat/clad the interior wall of the column (e.g., which comes intocontact with the sample and mobile phase in the fluidic path) with aninert material such as fused silica or PEEK. A preferred vacuum pressurethat may be used is described above such as at pressures below 10⁻³ atm.The particle size used for the column material may have a size less than2 microns, such as in the approximate inclusive range of 1.5-2 micronsize particles. It should be noted that techniques herein may also beused with larger size particles but that thermal effects become lessimportant for particles of larger sizes such as, for example, particlesexceeding the general size range of 5-10 microns, or greater than 5microns. Thus, thermal effects become more important for smaller sizedparticles. LC columns of any suitable dimension may be used inconnection with techniques herein. Exemplary dimensions for LC columnsthat may be used in an embodiment may have a length of 20 mm to 300 mm,and a diameter which is approximately equal to or more than 100 μm toabout 50 mm. As will be appreciated by those skilled in the art, thermaleffects may be insignificant for small diameter columns, for example,approximately less than 100 μm, as heat transfer will minimize radialand axial gradients. At larger diameters, the size of about 50 mm may bebased on practical limitations such as due to the pressure rating of thehardware. As diameter increases, it becomes significantly more expensiveto make a tube that can withstand the necessary high pressures.

Referring to FIG. 3, shown is an example of another embodiment of achromatography column in accordance with techniques herein. Theembodiment of FIG. 3 may be used as the column 110 in the system ofFIG. 1. FIG. 3 includes components similar to those of FIG. 2 with adifference that the insulation member or layer is denoted as 160 (ratherthan 130 as in FIG. 2) and is included as an integrated part or layer ofthe column. In the example 150, the column may be characterized asincluding a first inner portion 128 (the uninsulated column 128 asdescribed above in connection with FIG. 2) surrounded by a second outerportion forming the insulating layer 160. In the example 150 of FIG. 3,the insulating layer 160 may not extend the entire length of the innerportion 128. The insulating layer 160 may be formed as described abovein connection with element 130 of FIG. 2.

Referring to FIG. 4, shown is an example of another embodiment of achromatography column in accordance with techniques herein. Theembodiment of FIG. 4 may be used as the column 110 in the system ofFIG. 1. FIG. 4 includes components similar to those of FIG. 3 where theinsulation member or layer 160 is included as an integrated part orlayer of the column. In the example 180 (as also in FIG. 3), the columnmay be characterized as including a first inner portion 128 (asdescribed above in connection with FIG. 2) surrounded by a second outerportion forming the insulating layer 160. In the example 180 of FIG. 4,the insulating layer 160 may extend substantially the entire length ofthe inner portion 128. The insulating layer 160 may be formed asdescribed above in connection with element 130 of FIG. 2.

When performing experiments using LC systems, it may be desirable tosometimes heat or increase the temperature of the mobile phase to begreater than ambient or air temperature. To this end, further exemplaryembodiments are illustrated in connection with FIGS. 5A and 5B.

Referring to FIGS. 5A and 5B, shown are further example embodiments inwhich an active heating element may be positioned before the column toheat the mobile phase prior to the column inlet in accordance withtechniques herein.

FIG. 5A illustrates an example 200 of components of the LC system. Theexample 200 includes a pump 202, injector 204, heater 206 and encasementor housing 201 enclosing the LC column in a manner similar to that asdescribed above in connection with FIG. 2. The housing 201 may includethrough hole 250 having an exterior wall 220 functioning as the jacket120 of FIG. 2. Element 230 may be the chamber forming the insulatinglayer as described above in connection with element 130 of FIG. 2. Theheater 206 may be, for example, the ACQUITY Active Solvent heaterprovided by Waters Corporation. The heater 206 may, for example, be setat a desired set point temperature to heat the mobile phase having aflow path as represented by the arrows between the various components202, 204, 206 and 201.

FIG. 5B is a further variation to that of FIG. 5A. The example 280 ofFIG. 5B is similar to the example 200 of FIG. 5A with the differencethat the heater 206 is included within the housing 201 in FIG. 5B.

It should be noted that element 201 may be, for example, a column heatercompartment or oven into which the column is placed. The column heatermay include appropriate airtight seals as described herein to which avacuum pump may be connected (not shown) via through hole 250 asdescribed above in connection with through hole 50 of FIG. 2. In such anarrangement, the column heater may apply additional heating, as may beneeded, in addition to the active heating element represented by 206 forheating the mobile phase prior to entry into the column 128.Furthermore, an embodiment may use a column heater as described above inconnection with FIG. 5A with or without use of the solvent heater 206.The column heater (such as represented by 201 in FIGS. 5A and 5B) maybe, for example the Waters AQUITY UPLC® column heater.

In connection with the foregoing heater element 206 and/or embodimentwhere element 201 is a column heater, heating in connection withobtaining a desired set point may be performed using a feedback control(not illustrated) whereby the actual or observed temperature may beobtained, such as using one or more thermocouples, to provide feedbackto electronic controls of the heating components (e.g., toincrease/decrease amount of heat by controlling the heater based onwhether the current temperature measured by the thermocouple is at thedesired temperature set point, or within an acceptable threshold of sucha set point). In an embodiment using the column heater, the columnheater may be used to apply an additional source of heat to the columnenclosed within 201 in an airtight manner. The additional source of heatprovides for heating the column 128 via radiant heating from thesurrounding environment. It should be noted that other suitabletechniques may be used to also provide additional heat to the column 128included within the housing 201 with the above-mentioned insulatingmember or layer 230 such as formed by the vacuum chamber between theouter wall of the column 128 and the wall 220 of the housing 201. Forexample, an optional heater or heating means may enclose or surroundhousing 201 which provides the ability to add radiant heat to compensatefor potential non idealities in reaching true adiabatic conditions.

What will now be described are techniques that may be used in connectionwith performing an LC experiment during operation of an LC system toreduce the amount of time it takes to achieve steady state as describedabove with respect to measured axial thermal gradient whereby there willbe substantially constant values for column inlet temperature T_(in),column outlet temperature T_(out) and the difference (e.g., within someaccepted amount of measured threshold difference) between T_(in) andT_(out).

Techniques described in following paragraphs may use one or moreindependently controlled heaters in thermal contact with the column atvarious column positions. In some embodiments as described herein, aplurality of independently controlled heaters in thermal contact withthe column at various column positions may be utilized. Theseindependently controlled heaters may be used alone, or in combinationwith, other sources of heat that may be applied in connection withcontrolling the temperature of the column and liquid mobile phasepassing therethrough.

Temperature is one parameter that may have a significant effect on theretention of an analyte. Temperature may alter, for example, thekinetics of adsorption and desorption between an analyte and thestationary phase or separation medium thereby affecting both the speedand selectivity of the separation. In performing LC experiments, it isimportant that the LC column achieve a steady state temperature in orderto obtain reproducible results. Techniques described in followingparagraphs provide for reducing the time needed to achieve such steadystate with respect to column temperature. As described elsewhere herein,the steady state column temperature may be determined by having T_(in)and T_(out), and the difference therebetween (e.g., axial temperaturegradient) be relatively or substantially constant.

Referring to FIG. 6, shown is an example of an embodiment of componentsthat may be used in connection with the techniques herein for reducingthe amount of time required to achieve steady state as determined basedon substantially constant measured values for T_(in), T_(out) andtemperature difference between T_(in) and T_(out) (e.g., axial gradientthat is substantially constant). The example 300 includes a pump 202,injector 204, heater 206, and uninsulated column 128 having inlet 10 andoutlet 20 as described elsewhere herein. Additionally, the example 300includes a heater 310 coupled to, and in thermal contact with, thecolumn 128 at the column outlet 20. The column exit or outlettemperature T_(out) may be measured, for example, by a thermocouplepositioned at or near the column exit. Elements P1, P2 and P3 indicateexemplary positions where the thermocouple may be positioned to obtainthe temperature measurement T_(out). The heater 310 may be set, manuallyor through an automated control means (not illustrated), at a desiredset point temperature. The observed actual temperature may be measuredusing the thermocouple and the heater 310 may be turned on/off orotherwise adjusted to increase or decrease an amount of heat from theheater 310 based on whether the measured temperature T_(out) (e.g., suchas from one of P1, P2 or P3) is at the desired set point value. As knownin the art, a feedback technique such as using suitable electronicwiring, circuitry, and the like, may be used to automate control andoperation of the heater 310. For example with reference back to FIG. 1,code executing on the computer 116 may provide a user interface by whicha user may select and set a desired set point for T_(out). The measuredtemperature T_(out) may be provided to the computer system whereby codeexecuting on the computer system may compare the measured T_(out) to thedesired set point value for T_(out) and issue control signals to theheater 310 to appropriately control operation of the heater 310 inresponse to whether the measured T_(out) is at the desired set pointvalue for T_(out).

T_(in), the column inlet temperature as described above, may be measuredin a manner similar to that as T_(out). For example, a thermocouple maybe used to measure T_(in) at any suitable position at the column inletsuch as represented by P4, P5 and P6. Based on the observed or measuredvalues of T_(in) and T_(out) and determining a difference therebetween,an embodiment may determine when steady state has been reached asindicated by substantially constant measured values for T_(in), T_(out)and the axial thermal gradient or difference between T_(in) and T_(out).

The measurement of T_(in) and T_(out), control of the heater 310, andtemperature set point selection for T_(out) may be determined in usingany suitable manual and/or automated technique as will be appreciated bythose of ordinary skill in the art. For example, an embodiment may useautomated techniques such as described above using control signals tocontrol operation of the heater 310. Additionally, an embodiment mayalso determine the desired temperature set point for T_(out) in anautomated manner such as using an algorithm implemented by codeexecuting on a processor that predicts the steady state column exittemperature based on a given T_(in) and other parameters and uses thiscomputed value of T_(out) as the desired set point for T_(out). Thesteady state column exit temperature may be determined algorithmicallybased on the particulars of an implementation. For example, a predictedsteady state column exit temperature as a set point for T_(out) may bedetermined based on/using, column dimensions (e.g., length anddiameter), particle size, mobile phase composition (e.g., solvent), flowrate, column inlet temperature T_(in), and thermal properties of thecolumn assembly. For example, the following equation may be used inconnection with automated techniques to automatically predict a desiredT_(out) (predicted column exit temperature) as a set point for steadystate depending on the various parameters including a given T_(in). Forexample, an embodiment may use a PID (proportional-integral-derivative)controller to drive the set temperature of T_(out) to a steady statevalue based on a known or given T_(in) and other system parameters. Inthis manner, such techniques may be used to determine and drive thesystem to steady state. As known in the art, a PID controller may becharacterized as a generic control loop feedback mechanism (controller)as widely used in various type of control systems. The temperatureincrease in an adiabatic column that is heated via frictional heat canbe predicted by the Equation:

$\begin{matrix}{{\Delta\; T_{L}} = {\left( {1 - \overset{\_}{\alpha\; T}} \right)\frac{\Delta\; P}{\rho\; C_{p}}}} & {{EQUATION}\mspace{14mu} A}\end{matrix}$where ΔT_(L) is the longitudinal temperature difference between columninlet and outlet (e.g., T_(in)−T_(out)), α is the thermal expansioncoefficient of the mobile phase, T is the mean temperature of the mobilephase, ΔP is the pressure drop across the column, C_(p) is the heatcapacity of the mobile phase at constant pressure, and αT represents theaverage of a quantity αT, and ρ is the density of the mobile phase. Thevalue (1−αT) is on the order of ⅔ (F Gritti and G Guiochon, Anal. Chem.80 (2008) 5009). For example, using EQUATION A, an embodiment may useautomated techniques to determine a predicted value for T_(out)associated with steady state for a given or set T_(in). Appropriatecontrol signals may be sent to the heater/cooling unit 310 so thatT_(out) reaches and maintains (within some specified tolerance) itspredicted steady state value. As different values for T_(out) aredesired based on different given values for T_(in), such automatedtechniques may be used to determine and provide for adjusting T_(out)(e.g., by controlling 310). More generally, EQUATION A may be used todetermine particular pairs of values for T_(in) and T_(out) associatedwith a steady state. As described in more detail elsewhere herein, oneor more heating and/or cooling units may be used to drive T_(in) and/orT_(out) to desired temperatures as determined using EQUATION A.

A method for calculating the predicted steady state column exittemperature based on the foregoing may be implemented, for example,using software executing on a processor such as of the computer 116 ofFIG. 1. As described above, temperature control of the heater 310 may beobtained by adjusting or controlling the heater 310 via a feedback loopthat monitors the column exit temperature and sends control signals tothe heater 310 based on when a predicted desired set point for T_(out)has been reached (e.g., when the measured column exit temperatureT_(out) is at or near (such as within a threshold amount of) thepredicted value for T_(out)).

In connection with FIG. 6, it should be noted that the heater 206 may beoptionally utilized so that an embodiment in accordance with techniquesherein may only include heater 310 but not 206 as a solvent heater priorto entering the column 128. Additionally, in connection with FIG. 6,element 310 may be a heater or more generally a temperature control unitthat provides heating and/or cooling.

As a variation to the embodiment of FIG. 6, the unit represented by 310may be configured to be moveable or portable and readily positioned atother axial locations along the column 128 besides at the column exit oroutlet as illustrated.

Referring to FIG. 7, shown is an example of another embodiment ofcomponents that may be used in connection with the techniques herein forreducing the amount of time required to achieve steady state asdetermined based on substantially constant values for T_(in), T_(out)and temperature difference between T_(in) and T_(out) (e.g., axialgradient that is substantially constant). The example 400 includes apump 202, injector 204, heater 206, heater 310 and uninsulated column128 having inlet 10 and outlet 20 as described above in connection withFIG. 6. Additionally, the example 400 includes a second heater 410coupled to, and in thermal contact with, the column 128 at the columninlet 10. Element 410 may be similar to the heating and/or cooling unitrepresented by element 310 with the difference that 410 is located atthe inlet of column 128 functioning to control T_(in). Thus, in a mannersimilar to that as described above regarding T_(out), T_(in), may be setto a desired temperature set point and used as the set point forcontrolling heater 410. Heater 410 may be set, for example, via a manualand/or automated manner and the heater 410 may be controlled manually(e.g., user may turn on, off or otherwise adjust controls of the heater410 based on observed T_(in)) or automatically (e.g., using a feedbacktechnique with electronic temperature monitoring and control means toadjust the heater 410 based on observed or measured column inlettemperature T_(in) and a desired set point T_(in)). Such temperaturemonitoring and control means may include use of a computer or processorhaving code executing thereon which obtains observed temperatures fromtemperature sensors (e.g., such as located at any of P1-P6 of FIG. 6),and determines appropriate control signals sent over electroniccircuitry connected to appropriate ones of the heating/cooling ortemperature control units to effect a desired temperature adjustment.The observed temperature(s) may be used to determine what controlsignals, if any, are sent to one or more of the temperature controlunit(s) to achieve desired set point temperature(s) such as may bedetermined in accordance with EQUATION A.

Element 410 may be an independently controlled temperature control unitthat provides heating and/or cooling. For example, T_(in) may beselected as a desired set point and unit 410 may provide appropriateheating and/or cooling to achieve and maintain the desired set point forT_(in). Using T_(in) and the above-noted EQUATION A, a predicted desiredset point for T_(out) may be calculated and used as the desired setpoint for the unit 310. The units 310 and 410 may be independentlycontrolled to achieve and maintain a different desired set point foreach as may be used in an embodiment.

As another example of a use in connection with the embodiment 400 ofFIG. 7 when performing an LC experiment, the heater 206 may not beutilized. Additionally, the unit 410 may have a desired temperaturesetting T_(in) which is less than a desired temperature setting T_(out)of unit 310. Unit 410 may function as a cooling unit to reducetemperature of the mobile phase to be less than ambient temperature andunit 310 may function as a heating unit to increase the temperature ofthe mobile phase to be greater than ambient temperature and also greaterthan T_(in) (e.g., T_(in)<ambient temperature; T_(out)>ambienttemperature, and T_(in)<T_(out). As a further example, elements 410 and310 may be cooling units having desired set points both of which areless than ambient temperature and also where the set point T_(in) of 410is less than the set point T_(out) of 310 (e.g., T_(in)<ambienttemperature; T_(out)<ambient temperature, and T_(in)<T_(out)). As yet afurther example, elements 410 and 310 may be heating units havingdesired set points both of which are more than ambient temperature andalso where the set point T_(in) of 410 is less than the set pointT_(out) of 310 (e.g., T_(in)>ambient temperature; T_(out)>ambienttemperature, and T_(in)<T_(out)).

More generally, an embodiment in accordance with techniques herein mayinclude a plurality of heating and/or cooling units such as denoted by310, 410 above at any location along the column axis in thermal contactwith the column 128.

As will be appreciated by those skilled in the art and in connectionwith various examples described herein, EQUATION A may be used todetermine and predict a desired steady state value for T_(out) given aparticular set of parameters including T_(in). A heating/cooling unit310 may then be controlled to provide for appropriately adjustingT_(out) to be at a desired steady state predicted set point temperaturebased on EQUATION A. In a similar manner such as described in connectionwith FIG. 7, such techniques may be used to control operation of 410thereby driving or adjusting T_(in) to a desired predicted value such asbased on a particular given T_(out) value. More generally, the ΔT_(L) ofEQUATION A represents the temperature difference between twotemperatures in connection with steady state. As such, given EQUATION Aand one of the two temperatures used to compute ΔT_(L), the second ofthe two temperatures may be predicted. As described above, ΔT_(L), thetemperature difference, may be between T_(in) and T_(out) where eitherone may be known which is then used with EQUATION A to determine viacalculation the second temperature (e.g., T_(in) fixed or known and useEQUATION A to drive or determine T_(out). Alternatively, T_(out) may befixed or known and may be used to with EQUATION A to determine apredicted T_(in)). More generally, the automated techniques and EQUATIONA may be used with any two temperatures used to determine ΔT_(L) whereone of the two temperatures may be given and used to predict the othersecond temperature in connection with achieving a desired steady state.An embodiment may control operation of 310 and/or 410 based on desiredtemperatures for experimentation.

FIG. 8 is another example of such an embodiment. In the example 500, thecomponents similarly numbered may be as described above in connectionwith FIG. 7. Additionally, a third unit 510 providing heating and/orcooling may be positioned along the axis of column 128 in thermalcontact with the column 128. The units 310, 410 and/or 510 may besecured, coupled to, or more generally in thermal contact with, thecolumn 128. In one embodiment, the units 310, 410 and/or 510 may beattached or secured to the column 128 using clamps or other suitablemeans. For example, in one embodiment, ends of the column 128 may bepositioned within units represented by elements 310 and 410.

Based on the axial position or location on column 128 from the columninlet, element 510 may be considered as a unit for heating and/orcooling in a manner similar to 310 and/or 410. More generally, dependingon the axial location or position of 510 along the column, the desiredset point for 510 may be determined in accordance with EQUATION A. Thetemperature, T_(intermediate), at or near location 510 may be measuredusing temperature sensing devices as described herein in connection withmeasuring an observed value for T_(in) and/or T_(out). Similar means mayalso be used to control and adjust heating/cooling unit 510 as describedfor 310 and/or 410. A set point for T_(intermediate) may be determined,for example, based on a proportional temperature difference betweenT_(in) and T_(out) where such proportion is based on the distance orlocation of T_(intermediate) with respect to locations in the axialdirection along 128 of T_(in) and T_(out). For example, if 510T_(intermediate) is located midway or midpoint between T_(in) andT_(out) along 128, T_(intermediate) may be determined as approximatelyT_(in)+(½ ΔT_(L)) (e.g., also may be represented as (T_(in)+T_(out)/2).Thus, the desired set point temperature for T_(intermediate) may beestimated as a value between T_(in) and T_(out) which is proportional tothe location or distance of T_(intermediate) between the axial locationsalong column 128 where T_(in) and T_(out) are measured (e.g., columninlet and outlet).

The desired target or set point temperature of 510 may varyproportionally with the axial location of 510 on the column 128. Element510 may be associated with an intermediate temperature,T_(intermediate), and may be used in a manner similar to that asdescribed herein with T_(in) and/or T_(out). For example, any one ormore of 310, 410 and/or 510 may be adjusted in accordance with EQUATIONA to achieve steady state. For example, T_(in) may be known or given(whereby 410 may not be used or operated) and units 510 and/or 310 maybe controlled to achieve desired set point temperatures based onEQUATION A. T_(out) may be known or given (whereby 310 may not be usedor operated) and units 510 and/or 410 may be controlled to achievedesired set point temperatures based on EQUATION A. As another example,in connection with conditions for a particular experiment, set pointvalues for T_(in), T_(out) and T_(intermediate) may be determined basedon EQUATION A and units 410, 510 and/or 310 accordingly operated toachieve the desired set points values. Based on the above, anintermediate point T_(intermediate) may be located on the chromatographycolumn between the inlet and the outlet. The intermediate point of thechromatography column may be set to an intermediate temperature usingunit 510 (e.g, prior to injecting a sample in connection with obtainingsteady state). The intermediate temperature at the intermediate pointmay be between desired set point values for T_(in) and T_(out) (such asdetermined using EQUATION A). In one aspect, the desired intermediatetemperature set point may be determined as a sum of T_(in) and anapproximated value where the approximated value is a temperature offsetproportional to a distance of the intermediate point from the columninlet. In a similar manner, the desired intermediate temperature setpoint may be determined as a temperature offset with respect to T_(out).This temperature offset may be an approximated value proportional to adistance of the intermediate point from the outlet.

Thus, the example 500 of FIG. 8 illustrates one possible implementationof techniques herein using a plurality of units providing heating and/orcooling. By adding thermal energy (or otherwise more generally using oneor more auxiliary heating and/or cooling units as described herein) tovarious axial location points along a chromatographic column, thermalequilibrium may be obtained in less time than if the column were allowedto heat naturally via friction and other artifacts of the experimentproviding heat without use of the additional units such as 210, 310,410, and/or 510.

It should be noted that each of the exemplary embodiments of FIGS. 6, 7,and 8 as well as other embodiments such as described above (e.g., withadditional heaters in thermal contact with the column and/or placementof heaters in different axial positions along the column) may include aninsulating jacket surrounding the column and heaters.

As described above with reference again to FIG. 8, an embodiment may useelements 410, 510 and 310 in connection with, respectively, T_(in),T_(intermediate), and T_(out). As a variation to this, an embodimentmay, for example, omit use of T_(out) and 310 and rather include and useonly 410 and 510 respectively with T_(in) and T_(intermediate) inconnection with techniques herein. As yet another variation, anembodiment may, for example, omit use of T_(in) and 410 and ratherinclude and use only 510 and 310 respectively, with T_(intermediate) andT_(out) in connection with techniques herein.

As yet another variation, rather than have 510 represent aheating/cooling unit which may be controlled or adjusted, an embodimentmay alternatively just measure or monitor one or more intermediatetemperatures at one or more points between T_(in) and T_(out) alongcolumn 128 as part of feedback control processing. However, in thiscase, the one or more intermediate temperatures measured may be used tomodulate or control the units 410 and/or 310 based on a measuredintermediate temperature. The intermediate temperature(s) may be used asthe monitored or observed temperatures in connection with feedbacktechniques rather than use of measured or observed values for T_(in)and/or T_(out) at respective column endpoints. In this manner, anintermediate temperature may be used as a monitored or observedtemperature to adjust T_(in) and/or T_(out) by controlling operation of410 and/or 310 until the intermediate temperature observed is about atits desired set point value (obtained through adjustments made usingunits 410 and/or 310). To further illustrate, an embodiment may measureT_(intermediate) to determine whether the measured T_(intermediate) isat a desired value or set point. Accordingly, adjustments may be made tounit 410 until the measured T_(intermediate) is at its desired setpoint. Thus, T_(intermediate) may be used to control or adjust 410rather than make such adjustments to 410 based on a temperature measuredat T_(in) (endpoint of 128 at or near the location of 410 such asillustrated in FIG. 6).

Referring to FIG. 9, shown are examples of the embodiments from FIGS. 6,7 and 8 with the addition of a thermally insulating layer. Element 902may represent a thermally insulating jacket in each of 900, 910 and 920.The example 900 is an illustration of the column embodiment of FIG. 6 inthe surrounding jacket 902. The example 910 is an illustration of thecolumn embodiment of FIG. 7 in the surrounding jacket 902. The example920 is an illustration of the column embodiment of FIG. 6 in thesurrounding jacket 902. The jacket 902 may provide thermal insulation byreducing heat loss due to convective air currents. In one embodiment,the jacket 902 may provide sufficient insulation preventing thermalconductivity between the column (and its contents) and ambienttemperature such as of the environment outside of or surrounding thejacket 902.

The jacket 902 may be made from polystyrene foam (Styrofoam®), or moregenerally, any material exhibiting low thermal conductivity to act as aninsulating member. Polymers such as polymethacrylate, silicone,urethane, polyolefins, polyamide, polysulfone, polyethyramide,polycarbonate, rubber, polyester, polyfluoroelastomers and polyethyleneterephthalate, and the like, may also be used to form the jacket 902.Additionally, ceramics E.g., such as aerogels), fibrous materials (e.g.,such as methylcellulose and fiberglass) and the like, may also be usedto form the jacket 902. Although various thermal insulating materialshave been set forth in the foregoing illustrative embodiments as tomaterials that may be used to form jacket 902, any various suitablethermally insulting materials known in the art may be utilized. It willbe appreciated that such materials may be so shaped as to insulate thearea around the chromatography column to create a controlled air spaceor chamber so as to prevent or minimize a radial thermal gradient withinthe column. Furthermore, although such materials may be illustrated asimmediately surrounding the column, such materials may also beintegrated into the column itself, for example, such as used to form thecolumn outer walls.

As another variation, the jacket 902 may be made of steel or metal asdescribed above in connection with FIG. 2 so that the insulating layeror member is not the jacket 902 itself but rather the airspace 903surrounding the uninsulated column 128 (e.g., between the column 128 andthe surrounding jacket 902). In this case, element 903 may be thechamber or space, such as the vacuum chamber, forming the insulatinglayer and may be formed using any of the techniques, gases (e.g., insertgas, atmospheric gas), and the like, as described elsewhere herein suchas in connection with FIG. 2. In one embodiment, the chamber or airspace903 between the column 128 and the jacket 902 (e.g., surrounding thecolumn 128) may be at ambient pressure and aerogel particles may beincluded in the area 903 to provide insulation. Alternatively, theairspace 903 including the aerogel particular may be form a vacuumchamber having a pressure less than ambient pressure. Examples of suchpressures are described elsewhere herein. As a variation to the area 903including aerogel particles, the column 128 may be placed in a moldedaerogel component. The molded aerogel may surround the column and may beformed, for example, from two separately molded halves or portionswhich, when placed together, form a desired cavity approximating theshape of the column. The foregoing two molded aerogel portions may befitted together as part of assembly with the column inserted into theformed cavity. The foregoing embodiments using molded aerogel or aerogelparticles in a chamber may be used as an means of insulation inconnection with any of the embodiments of the column described hereinsuch as, for example, using one or more additional heating/coolingunits.

Referring to FIG. 10, shown are examples of the embodiments from FIGS.6, 7 and 8 with the addition of a thermally insulating layer 1002. Inthe examples 1000, 1010 and 1020 of FIG. 10, the insulating layer 1002may be an integral component of the column 128 such as describedelsewhere herein in connection with FIGS. 3 and 4. In the examples 1000,1010 and 1020, the insulating layer 1002 may be formed from any of thematerials of the jacket 902 as described above. As a further variation,the insulating layer 1002 may be formed as a layer between a surroundingouter jacket 902 forming the outer walls of the combination of thecolumn 128 and insulating layer 1002. The jacket 902 in this case may bemade of steel, titanium, or other suitable material such as describedelsewhere herein in connection with FIG. 2 and the insulating layer 1002may be the chamber or space, such as the vacuum chamber, forming theinsulating layer using any of the techniques, gases (e.g., insert gas,atmospheric gas), and the like, as described elsewhere herein such as inconnection with FIG. 2 (and above in FIG. 9).

In connection with the example 1020, it should be noted that the unit510 that performs heating and/or cooling should have sufficient thermalcontact with the uninsulated column 128. For example, the insulatinglayer 1002 is illustrated as being formed around the unit 510 in areasbounded by or between, for example, units 410 and 510 and bounded by, orbetween, units 510 and 310.

In connection with embodiments described herein, such as with FIGS. 9and 10, utilizing an insulating layer and/or jacket, it will beappreciated by those of ordinary skill in the art that the column inlettemperature, T_(in) and column outlet temperature, T_(out), may bemeasured by placement of various thermocouples in thermal contact withthe uninsulated column 128 such as between any insulating layer and thecolumn 128.

Axial control of column temperature as may be achieved using embodimentsdescribed herein such as in connection with FIGS. 6-10 may provideadditional benefits. For example, axial control of temperature such asby control and selection of T_(in) and T_(out) may facilitatereproducibility of experimental conditions and chromatographic methods.Such techniques may provide for reproducibility of experimentalconditions using columns having similar properties as well as differentproperties. For example, a typical HPLC column with a mean particle sizeof 5 microns generates less heat than a column of equivalent dimensionsusing 1.7 micron size particles. Without use of the techniques herein,the resulting thermal gradient during experiments may be different onthe two columns resulting in differences in experimental data obtainedusing the two columns. The techniques herein may be used with the twocolumns—each using a different size particle—to create identical thermalgradients for the two columns.

As another example, one way to increase throughput in chromatography isto operate at a faster flow rate. It may not be desired or expected tohave an increased flow rate affect chromatographic selectivity. However,the thermal gradients across two columns would not be identical wherethe two columns have the same properties (e.g., dimensions, particlesize, etc. affecting the experiment) and where each of the two columnshas a different flow rate (e.g., since the frictional heat generatedvaries with, and is directly proportional to, the flow rate of themobile phase). In experiments where the chromatographic selectivity(e.g., distance between peaks of eluting analytes) is altered with thechanging flow rate, the axial thermal gradient may be altered so thatthe two experiments using the different flow rates provide similarselectivity. As known in the art, chromatographic selectivity (alsoreferred to as a separation factor or relative retention ratio) is ameasure of the time or distance between the maxima of two peaks. Thechromatographic selectivity may be represented as K2/K1 where K1 is theretention factor of the first peak and K2 is the retention factor of thesecond peak. If K2/K1=1, then the peaks have the same retention andco-elute.

As another advantage, adding one or more independently controlledheaters along the column body such as described above may reduce overallcosts when compared to other alternatives such as use of a columnheater.

In connection with embodiments as described herein such as using avacuum insulating layer or chamber surrounding the LC column, trueadiabatic conditions may be approached to minimize radial thermalgradients and eliminate or minimize convective heat loss.

In connection with techniques herein, the inventors performedexperiments that will now be described. A 2.1×100 mm Waters ACQUITY BEHC18 1.7 μm column was connected to a Waters ACQUITY™ UPLC instrument.0.5 microliters of a sample containing the following 5 components: (1)0.046 mg/mL thiourea; (2) 0.080 mg/mL dodecanophenone; (3) 0.1 mg/mLtetradecanophenone; (4) 0.10 mg/mL hexadecanophenone; and (5) 0.483mg/mL di-n-decyl phthalate, was injected onto the column using a mobilephase of acetonitrile. Analyses were made using the following flowrates: 0.45, 0.50, 0.55, 0.65, 0.75, 0.85, 0.95, 1.05, 1.10, 1.15, and1.20 mL/minute. The column was thermally equilibrated between changes inflow rate by monitoring repeat injections of the test probes untilretention time reproducibility was achieved. Detection was by UV at 240nm.

The vacuum system used in the experiment was a Pfeiffer Vacuum TSH 071ETurbomolecular Drag Pumping Station, which included the followingstandard components: a Pfieffer-Balzers TMH-071P Turbomolecular DragPump, with DN-63-ISO inlet flange and comes standard with a solid-statefrequency converter, and electronic controls. A dual-stage,high-performance rotary vane pump with a pumping speed of 2.5 m³/h fromPfeiffer (Duo 2.5 model PKD41707) was used to bring the vacuum quicklyto ˜10⁻² torr before starting the turbo pump. The vacuum chamber for thecolumn and the connections to the vacuum system were constructed fromMDC Vacuum Products (Hayward, Calif.) 304 stainless steel tubing andseals (Viton® or Buna-N® O-rings) rated to 10⁻⁸ torr. For vacuum readingbetween atmosphere to 10⁻² mbar/Torr an Edwards active pirani gauge partnumber D02177000 APG-1-NW16 ST/ST was used. For vacuum readings between10⁻³ to 10⁻⁸ mbar/Torr an Edwards active Inverted Magnetron Gauge partnumber D14641000 AIM-S-NW25 was used. The vacuum readings were taken asclose (˜4″ away) from the column vacuum chamber.

Chromatographic performance was evaluated for columns under fourdifferent environments: (A) Isothermal: The column was placed in are-circulating water bath (RTE-111, Thermo NESLAB) maintained at 25 C;(B) Still air: The column was placed inside a box of approximately20″×33″×34″ to minimize convection in the surrounding air; (C) Insulatedwith aerogel: Column was placed inside a chamber filled with granularaerogel from United Nuclear Scientific (Laingsburg, Mich.); (D) Thecolumn was jacketed in a vacuum of 3×10⁻⁵ torr using a roughing pump anda diffusion pump.

Referring to FIG. 11, shown are chromatogram of the resultingseparations for the above-mentioned four testing environments (A)-(D).The example 1100 includes 4 sets of chromatographic data obtained for aflow rate of 1.2 mL/minute. Element 1120 represents the chromatogramobtained for testing environment condition (A) for isothermal conditionswhere the column was placed in a re-circulating water bath (RTE-111,Thermo NESLAB) maintained at 25 C. Element 1140 represents thechromatogram obtained for testing environment condition (B) using stillair where the column was placed inside a box of approximately20″×33″×34″ to minimize convection in the surrounding air. Element 1160represents the chromatogram obtained for testing environment condition(C) where insulation was provided using aerogel where, as noted above,the column was placed inside a chamber filled with granular aerogel fromUnited Nuclear Scientific (Laingsburg, Mich.). Element 1180 representsthe chromatogram obtained for testing environment condition (D) wherebythe column was jacketed in a vacuum of 3×10⁻⁵ torr using a roughing pumpand a diffusion pump. In connection with the chromatograms of 1100, theX axis of each denotes time, in minutes. In this example, the detectorwas a UV absorbance detector so that the detection units on the Y axisrepresent absorbance at 240 nm (AU). Each of the chromatograms 1120,1140, 1160 and 1180 includes 5 peaks denoted 1-5 which respectivelycorrespond to peaks for the 5 components of the sample as describedabove.

Shown below is a table illustrating the plate count for Peak (4)corresponding to hexanophenone, at the different flow rates. Resultsshow that as flow rate increases, the plate count is highest in thecases where the column is at near adiabatic conditions.

Flow Rate (A) (B) (C) (D) (mL/min) Isothermal Still air Insulated Vacuum0.45 25,536 25,045 27,160 27,227 0.50 22,598 22,829 26,108 26,892 0.5519,175 20,908 24,956 26,382 0.65 12,733 16,954 22,756 25,518 0.75 7,99713,492 20,747 23,607 0.85 5,099 10,866 19,014 22,104 0.95 3,389 8,92617,386 20,489 1.05 2,357 7,386 15,875 18,898 1.15 1,715 6,259 14,56717,098 1.20 1,478 5,839 14,095 16,371

Variations, modifications, and other implementations of what isdescribed herein will occur to those of ordinary skill in the artwithout departing from the spirit and the scope of the invention asclaimed. Accordingly, the invention is to be defined not by thepreceding illustrative description but instead by the spirit and scopeof the following claims.

What is claimed is:
 1. An apparatus for liquid chromatography,comprising: a liquid chromatography column having an inlet for receivinga mobile phase and an outlet for the mobile phase to exit the column; aheater positioned to be in thermal contact with the liquidchromatography column; an outlet temperature sensor for measuringtemperature of the mobile phase exiting the liquid chromatography columnat the outlet; and a processor configured to: determined a desired setpoint for the temperature of the mobile phase exiting the liquidchromatography column at the outlet to accelerate thermal equilibriumfor the liquid chromatography column by predicting a steady statetemperature of the mobile phase at the outlet when the thermalequilibrium is reached and setting the steady state temperature as thedesired set point; receive the measured temperature of the mobile phaseexiting the liquid chromatography column at the outlet from the outlettemperature sensor; determine a difference between the received measuredtemperature of the mobile phase exiting the liquid chromatography columnat the outlet and the desired set point; and based on the determineddifference, control the heater to impart heat to the mobile phase toattempt to reach the desired set point.
 2. The apparatus of claim 1,wherein the heater is positioned in proximity to the outlet of theliquid chromatography column.
 3. The apparatus of claim 1, wherein thecontrolling the heater to impart heat to the mobile phase to attempt toreach the desired set point comprises controlling the heater to impart adesignated amount of heat.
 4. The apparatus of claim 1, wherein theapparatus further comprises an inlet temperature sensor for measuring atemperature of the mobile phase entering the liquid chromatographycolumn at the inlet and wherein the processor predicts the steady statetemperature for the temperature of the mobile phase exiting the liquidchromatography column at the outlet based at least in part on thetemperature of the mobile phase entering the liquid chromatographycolumn at the inlet sensed by the inlet temperature sensor.
 5. Theapparatus of claim 4, wherein the processor determines the desired setpoint for the temperature of the mobile phase exiting the liquidchromatography column at the outlet to keep a difference between thetemperature of the mobile phase exiting the liquid chromatography columnat the outlet and the temperature of the mobile phase entering theliquid chromatography column at the inlet substantially constant.
 6. Theapparatus of claim 5, wherein the processor determines a predicteddifference between the temperature of the mobile phase exiting theliquid chromatography column at the outlet and the temperature of themobile phase entering the liquid chromatography column at the inlet, andwherein, given a temperature sensor reading of the temperature of themobile phase entering the liquid chromatography column at the inlet, theprocessor predicts the steady state temperature of the mobile phaseexiting the liquid chromatography column at the outlet.
 7. The apparatusof claim 6, wherein the processor determines the predicted differencebetween the temperature of the mobile phase exiting the liquidchromatography column at the outlet and the temperature of the mobilephase entering the liquid chromatography column at the inlet based on athermal coefficient of the mobile phase, a mean temperature of themobile phase, a density of the mobile phase, heat capacity of the mobilephase and pressure drop across the liquid chromatography column.
 8. Theapparatus of claim 1, wherein the processor predicts the steady statetemperature of the mobile phase exiting the liquid chromatography columnat the outlet based at least in part on a length and a diameter of theliquid chromatography column.
 9. The apparatus of claim 1, wherein theprocessor predicts the steady state temperature of the mobile phaseexiting the liquid chromatography column at the outlet based at least inpart on a size of particles in the liquid chromatography column.
 10. Theapparatus of claim 1, wherein the processor predicts the steady statetemperature of the mobile phase exiting the liquid chromatography columnat the outlet based at least in part on a flow rate of the mobile phasethrough the liquid chromatography column.
 11. The apparatus of claim 1,wherein the processor predicts the steady state temperature of themobile phase exiting the liquid chromatography column at the outletbased at least in part on a composition of the mobile phase.
 12. Theapparatus of claim 11, wherein the processor predicts the steady statetemperature of the mobile phase exiting the liquid chromatography columnat the outlet based at least in part on a solvent in the mobile phase.13. A system, comprising: a storage for storing programming instructionsand data; and a processor configured to: determine a desired set pointfor a temperature of a mobile phase exiting the liquid chromatographycolumn at an outlet to accelerate thermal equilibrium for the liquidchromatography column by predicting a steady state temperature of themobile phase at the outlet when the thermal equilibrium is reached andsetting the steady state temperature as the desired set point; receive ameasured temperature of the mobile phase exiting the liquidchromatography column at the outlet from an outlet temperature sensor;determine a difference between the received measured temperature of themobile phase exiting the liquid chromatography column at the outlet andthe desired set point; and based on the determined difference, controlthe heater to impart heat to the mobile phase to attempt to reach thedesired set point.
 14. The system of claim 13, wherein the heater ispositioned in proximity to the outlet of the liquid chromatographycolumn.
 15. The system of claim 13, wherein the controlling the heaterto impart heat to the mobile phase to attempt to reach the desired setpoint comprises controlling the heater to impart a designated amount ofheat.
 16. The system of claim 13, wherein the processor predicts thesteady state temperature of the mobile phase exiting the liquidchromatography column at the outlet based at least in part on atemperature of the mobile phase entering the liquid chromatographycolumn at an inlet that is sensed by a temperature sensor and providedto the processor.
 17. The system of claim 16, wherein the processordetermines the desired set point for the temperature of the mobile phaseexiting the liquid chromatography column at the outlet to keep adifference between the temperature of the mobile phase exiting theliquid chromatography column at the outlet and the temperature of themobile phase entering the liquid chromatography column at the inletsubstantially constant.
 18. The system of claim 17, wherein theprocessor determines a predicted difference between the temperature ofthe mobile phase exiting the liquid chromatography column at the outletand the temperature of the mobile phase entering the liquidchromatography column at the inlet, and the processor, given atemperature sensor reading of the temperature of the mobile phaseentering the liquid chromatography column at the inlet that is sensed bythe temperature sensor and provided to the processor, determines thesteady state temperature of the mobile phase exiting the liquidchromatography column at the outlet.
 19. The system of claim 18, whereinthe processor determines the predicted difference between thetemperature of the mobile phase exiting the liquid chromatography columnat the outlet and the temperature of the mobile phase entering theliquid chromatography column at the inlet based on a thermal coefficientof the mobile phase, a mean temperature of the mobile phase, a densityof the mobile phase, heat capacity of the mobile phase and pressure dropacross the liquid chromatography column.
 20. The system of claim 13,wherein the processor predicts the steady state temperature of themobile phase exiting the liquid chromatography column at the outletbased at least in part on a length and a diameter of the liquidchromatography column.
 21. The system of claim 13, wherein the processorpredicts the steady state temperature of the mobile phase exiting theliquid chromatography column at the outlet based at least in part on asize of particles in the liquid chromatography column.
 22. The system ofclaim 13, wherein the processor predicts the steady state temperature ofthe mobile phase exiting the liquid chromatography column at the outletbased at least in part on a flow rate of the mobile phase through theliquid chromatography column.
 23. The system of claim 13, wherein theprocessor predicts the temperature of the mobile phase exiting theliquid chromatography column at the outlet based at least in part on acomposition of the mobile phase.
 24. The system of claim 13, wherein theprocessor predicts the temperature of the mobile phase exiting theliquid chromatography column at the outlet based at least in part on asolvent in the mobile phase.
 25. A method performed by a processor,comprising: determining a desired set point for a temperature of amobile phase exiting a liquid chromatography column at an outlet toaccelerate thermal equilibrium for the liquid chromatography column bypredicting a steady state temperature of the mobile phase at the outletwhen the thermal equilibrium is reached and setting the steady statetemperature as the desired set point; receiving a measured temperatureof the mobile phase exiting the liquid chromatography column at theoutlet from an outlet temperature sensor; determining a differencebetween the received measured temperature of the mobile phase exitingthe liquid chromatography column at the outlet and the desired setpoint; and based on the determined difference, controlling a heater toimpart heat to the mobile phase to attempt to reach the desired setpoint.